This application is a divisional of U.S. patent application Ser. No. 08/964,162, now abandoned, filed on Nov. 4, 1997, which is a continuing application of U.S. Provisional Application Ser. No. 60/030,302, filed Nov. 5, 1996, the content of which is specifically incorporated herein by reference in its entirety.
The United States government has rights in the present invention pursuant to grant number HL-45851 from the National Institutes of Health.
1.1 Field of the Invention
The present invention relates generally to the field of molecular biology. Disclosed are methods and compositions comprising DNA segments encoding a novel protein, sentrin-1, found to protect against TNF and Fas/APO-1 induced apoptosis. Various methods for making and using these DNA segments, DNA segments encoding synthetically-modified sentrin-1 polypeptides, and native and synthetic sentrin-1 peptides are disclosed, such as, for example, the use of DNA segments as diagnostic probes and templates for protein production, and the use of proteins, fusion protein carriers and peptides in various immunological and diagnostic applications.
1.2 Description of Related Art
Fas/APO-1 (CD95), belongs to the TNF receptor superfamily, which is characterized by cysteine-rich pseudo-repeats in the extracellular domain (Smith et al., 1994). Despite the similarity in the organization of the extracellular domain, the cytoplasmic domain of the TNF receptor superfamily is not conserved implying that different signaling mechanisms must be operative for different receptors. Nonetheless, Fas/APO-1 and TNFR1 share a common cytoplasmic signaling motif called the “death domain” (Itoh et al., 1991; Itoh and Nagata, 1993; Tartaglia et al., 1993). Deletion or mutation in the death domain abolishes the ability of these receptors to transduce an apoptosis signal.
Several laboratories have reported the cloning of death-domain associated proteins, including FADD/MORT1, TRADD, and RIP (Boldin et al., 1995; Chinnaiyan et al., 1995; Hsu et al., 1995; Stanger et al., 1995). Following ligation of Fas on the cell surface by either antibody or ligand, a complex called DISC (Death-Inducing Signal Complex), which include Fas, FADD/MORT1 and FLICE/MACH, is formed via death-domain or death-effector domain-mediated interaction (Kischkel et al., 1995; Muzio et al., 1996; Boldin et al., 1996). Ligand-induced association of TNFR1 with TRADD, RIP, and FADD/MORt1 has also been demonstrated (Hsu et al., 1995; Hsu et al., 1996a; Hsu et al., 1996b). Taken together, death-domain/death domain interactions initiate a platform for the assembly of signaling complexes which are essential for apoptosis induction or NF-kB signaling.
Several non-death-domain containing proteins which bind to either Fas/APO-1 or TNFR1 have also been reported. FAP-1 is a tyrosine phosphatase which binds to the C-terminal 15 amino acids of Fas/APO-1, a negative regulator of cell death signaling (Sato et al., 1995). Overexpression of FAP-1 could inhibit Fas/APO-1 signaling. FAF1, another novel protein that binds to the Fas/APO-1 cell death domain, facilitates Fas/APO-1-mediated apoptosis (Chu et al., 1995). Furthermore, there are a large number of proteins which regulate apoptosis either positively or negatively, but do not bind to the cytoplasmic domain of either Fas/APO-1 or TNFR1. They include the IAP's (Liston et al., 1996), ALG (Vito et al., 1996), members of the Bcl-2 family (Oltvai and Korsmeyer, 1994), and inhibitors of the ICE family, such as CrmA and P35 (Clem and Miller, 1994). Full integration of these proteins in the cell death signaling pathway is yet to be achieved. However, the functions of these proteins are yet to be completely elucidated and their interrelationships remain undefined.
PML, a RING finger protein with tumor suppressor activity, has been implicated in the pathogenesis of acute promyelocytic leukemia that arises following a reciprocal chromosomal translocation that fuses the PML gene with the retinoic acid receptor α (RARα) gene. Immunocytochemical analysis has demonstrated that PML is co-localized with a novel ubiquitin-like protein in the nuclear bodies, which could be disrupted by the PML-RARα fusion protein. The physical nature of this co-localization is unknown. Using a COS cell expression system, the inventors show that PML is covalently modified by the sentrin family of ubiquitin like proteins, but not by NEDD8 or ubiquitin.
Chromosomal translocation (Larson et al., 1984; Lapenta et al., 1997), detected in the majority of patients with acute promyelocytic leukemia, generates a fusion protein composed of portions of the retinoic acid receptor α (RARα) and RING finger protein called PML (Larson et al., 1984; de The et al., 1991; Kakizuka et al., 1991; Kastner et al., 1992). In cell lines derived from patients with acute promyelocytic leukemia, the nuclear bodies are disrupted into a microparticulate pattern, which is reversible by treatment with retinoic acid (Weis et al., 1994; Dyck et al., Cell, 1994). PML has also been shown to suppress the transformation of NIH3T3 cells by the activated neu oncogene (Liu et al., 1995). Using full length PML as bait in a yeast two hybrid interaction screening, Boddy et al. (1996) have isolated a novel ubiquitin-like protein, called PIC1 that interacted specifically with PML in the yeast interaction assay (Boddy et al., 1996). They have further shown that PIC1 was co-localized with PML to the nuclear bodies. In NB4 cells, which are derived from acute promyelocytic leukemia, there was no significant co-localization of PIC1 with PML. However, following retinoic acid treatment, a significant re-localization of PIC1 with PML was observed. These observations suggest that the association of PIC1 with PML may play an important role in the pathogenesis of acute promyelocytic leukemia.
Protein modification by ubiquitin is critical for targeting proteins to be degraded by proteasomes (Coux et al., 1996; Hershko and Ciechanover, 1992; Jentsch, 1992). Conjugation of ubiquitin to other proteins requires initial activation of the conserved C-terminal Gly residue catalyzed by a specific ubiquitin-activating enzyme, E1. An intermediate, ubiquitin adenylate, is formed by displacement of PPi from ATP. Ubiquitin adenylate is then transferred to a thiol site in E1 with release of AMP. Next, ubiquitin is transferred to a family of ubiquitin-carrier proteins, E2, through transacylation. Finally, ubiquitin is transferred from E2 to its target protein through an isopeptide linkage with the ε-amino group of the Lys residue of the target protein. The transfer of ubiquitin from E2 to the target protein may require the participation of a ligase, E3. The internal Lys of ubiquitin, in particular Lys48, can also be modified by another ubiquitin to form multiubiquitin chains which may be crucial for proteosome recognition (Finley et al., 1994). In recent years, ubiquitination has been shown to play a critical role in antigen processing, in the regulation of cell cycle, in receptor endocytosis, and in signal transduction (Hochstrasser, 1996; Rock et al., 1994; Murray, 1995).
Ubiquitin is not the only molecular tag for protein modification. Another ubiquitin-like protein, UCRP, has been shown to be conjugated to a large number of intracellular proteins (Haas et al., 1987). UCRP contains two ubiquitin domains and is inducible by type 1 interferons. There is evidence for a distinct pathway of UCRP conjugation that is parallel to ubiquitination (Narasimhan et al., 1996).